The mammalian intestinal epithelium is a continuously developing system. Each intestinal crypt contains a single, multipotent stem cell which undergoes rapid proliferation. Descendants of this stem cell become the four principal cell types that constitute the gut epithelium: absorptive, goblet, enteroendocrine, and Paneth cells. This coupled cell proliferation and differentiation is subject to stringent regulation. Expression of many intestinal genes follow a gradient of distribution along the duodenal-colon (horizontal) axis, and the crypt-villus and crypt-surface epithelium (vertical axes of the small and large intestines, respectively. The unique region- specific, cell-specific, and spatial-specific nature of gene regulation renders the intestinal epithelium attractive for studying the mechanism controlling cell proliferation and differentiation. We previously isolated a gene, named A4, that is transcriptionally activated upon differentiation of the human colonic epithelial cell line HT29-18. Expression of A4 is enriched in the intestinal epithelium and follows an increasing, horizontal gradient from the small to the large intestine. In addition, expression of A4 is abundant in the crypt and follows a diminishing, vertical gradient along the crypt-villus and crypt-surface epithelium axIs of the small and large intestine, respectively. These salient features suggest that A4 may serve as an excellent marker of crypt cell differentiation, and that understanding the mechanism regulating A4 expression in vitro may shed some light on the mechanism controlling gut epithelial differentiation. Within the project period of the current Program Project, we have made significant progress in understanding the regulation of the A4 promoter during HT29 differentiation. Specifically, we identified a positive cis-element in the proximal A4 promoter that interacts with a DNA-binding protein (putative transcription factor) with a differentiation- dependent activity. In addition, we showed that A4 is a member of the proteolipid family of proteins and is a membrane protein of the endoplasmic reticulum with putative ion channel activity. Two Specific Aims are proposed in the competitive renewal application for Project 5: (1) to isolate and characterize the differentiation- dependent DNA-binding factor (named GATG-binding protein) that interacts with the A4 promoter and to study its effect on epithelial differentiation, and (2) to further characterize the function of A4 as an ion channel and as a potential mediator of differentiation. These studies should help understand the mechanism controlling intestinal epithelial differentiation.